If Chlamydia trachomatis is highly suspected, as it is a common STD that can cause UTI, some portion of the urine sample can be send for DNA based analysis method such as polymerase chain reaction(PCR).

Case study 2

Besides Salmonella, there are also several other possible microorganisms that could lead to enterocolitis or cause the diarrhea in the patient. Here are the other possibilities:1. Enterotoxigenic Escherichia coli2. Campylobacter jejuni3. Clostridium difficileShigella: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei

The female patient in case 1 was diagnosed with urinary tract infection. UTI occurs due to the infection of microorganisms in the bladder,urethra and kidneys. It can be further classified as cystitis( bladder infection) or pyelonephritis( kidney infection). The patient showed common UTI symptoms seen in cystitis like dysuria(painful urination), fever and chills, but did not complain of back pain, or haematuria, hence it is likely that she is suffering from cystitis.

UTI occurs more frequently in woman as compared to men, due to the short urethra they have, allowing entry of bacteria into the urinary tract. The most common cause of UTI is Escherichia coli, a normal flora in the intestine and colon that enters and invades the urethra causing an infection. The second most likely bacteria that can cause UTI is Staphylococcus saprophyticus, as it usually infects woman in between the age of 20-40. Other microbes such as Klebsiella pneumoniae, Proteus mirabilis and Enterococcus species can also cause UTI, however, their occurrence is very low .

They are eliminated because:a) Enterocoocus species usually occurs in patients who have undergone urinary tract surgery.b) Proteus mirabilis often infect recurrent UTI patients that have structural abnormalities in their urinary system.c) Klebsiella pneumoniae is involved in hospital-acquired infection and commonly infect immunocomprised individuals.

Most possible diagnosis: Escherichia coli and Staphylococcus saprophyticusAll the above characteristics were not observed in the patient and hence can be eliminated.The two most likely cause of UTI are Escherichia coli and Staphylococcus saprophyticus.

Types of enterocolitis:1) Necrotizing enterocolitis●Gastrointestinal disease that mostly affects premature infants, NEC involves infection and inflammation that causes destruction of the bowel intestine or part of the bowel.● NEC typically occurs within the first 2 weeks of life, usually after milk feeding has begun 2) Autistic enterocolitis●Autistic enterocolitis is a controversial term first used by British gastroenterologist Andrew Wakefield to describe a number of common clinical symptoms and signs which he contends are distinctive to autism.● The existence of autistic enterocolitis is controversial, as the methodology of Wakefield's studies has been criticized and his results have not been replicated by other groups

3) Salmonella enterocolitis● Most common type of food poisoning● Infection in the lining of the small intestine caused by the bacteria Salmonella.●Symptoms include diarrhea and abdominal pain

Most possible diagnosis: Salmonella enterocolitisReasons: Since necrotizing enterocolitis affects mainly infants but the patient is a 29-year-old female, it is highly unlikely that she is suffering from this disease. Moreover, it is not known to the medical technologist that the patient is suffering from autism. Hence, there is very low chance of her suffering from autism enterocolitis. The symptoms of salmonella enterocolitis include diarrhea which is one of the complaints as told by the female patient.

Indwelling cathetersIndwelling catheters avoid distension by emptying the bladder continuously into a bedside drainage collector. Individuals with indwelling catheters are encouraged to maintain a high fluid intake in order to prevent bacteria from accumulating and growing in the urine.

Possible agents:● Many different Gram-negative organisms colonize urinary catheters, often becoming invasive infections.●The most commonly isolated pathogens are Escherichia coli and Enterococcus spp.E.coli uses fimbriae to adhere to the urinary epithelium, thereby reducing the risk of being washed away.● Infections caused by Proteus spp. are more likely in patients who have stones as Proteus spp. have urease activity that raises urinary pH, thus encouraging stone formation.● Staphylococcus saprophyticus is a common isolate from sexually active females.● Other intestinal bacteria, including Klebsiella pneumoniae (K.pneumoniae), Proteus mirabilis (P.mirabilis) , and Citrobacter.●Others include Pseudomonas aeruginosa (P.aeruginosa), Enterobacter, and Serratia species, gram-positive organisms, including Enterococcus species, and S. saprophyticus .

Most possible diagnosis: Staphylococcus saprophyticus, Klebsiella pneumoniae, Proteus mirabilis and EnterococcusUTI in this patient should be due to the presence of the catheter in the urethra. Hence these microbes might the possible reason.

Information on BronchitisInflammation of the mucous membrane in the lungs' bronchial passagesNarrowed bronchial passages shuts off the tiny airways in the lungsResults in coughing spells, thick phlegm and breathlessnessTwo forms: acute (lasts less than 6 weeks) and chronic (more than two years)

Acute bronchitis●responsible for the hacking cough and phlegm production that sometimes accompany an upper respiratory infection● In most cases the infection is viral in origin, but sometimes it's caused by bacteria● very common among both children and adults

Possible agents1) Adenovirus● Non-enveloped double-stranded linear DNA● Icosahedral nucleocapsid with a fiber protruding from each of the 12 vertices●Causes bronchitis when it affects the lower respiratory tract

Wound Infection caused by bacteria● This is because swelling is one of the hallmarks of inflammation due to infection by either endogenous factors like tissue necrosis or exogenous factors like microorganism infections.● Fever, on the other hand, is a common manifestation of infection and inflammation that is caused by many bacterial products eg endogenous or exogenous pyrogens.● Most wound infections are caused by normal flora found on the skin/body.

Most possible microbes:However, out of all the organisms, the most common bacteria involved in wound infection due to operation was found to be Staphylococcus aureus, which accounts for 17-20% of the cases reported. Out of these cases, 40-50% are due to MRSA (Methicillin-resistant Staphylococcus aureus).

2) Bacterial VaginosisIt is caused by imbalance of bacteria flora in vagina. Usually, it is characteristic by the overgrowth of Gardnerella vaginalis and Gardneralla mobiluncus,.Gardnerella bacteria is facultative anaerobic and gram-negative, while Mycoplasma hominisIt symptoms includes gray vaginal discharge and painful urination.

Tuesday, November 6, 2007

hey people,im really sorry for the super late posting..for this posting i will share with u guys the quality assurance and quality control in the histopathological laboratory where im attached to.

Quality Assurance(QA) and Quality Control(QC)QA is a program in which overall activities conducted by the institution are directed towards assuring the quality of the products and services provided. QC is under QA in which products and services provided are evaluated in hopes of achieving high accuracy and reliability of test results and also aids troubleshooting. This involves continuous monitoring of operations and systemic day-today checking of the produced data to decide whether these are reliable enough to be released.

Application of QC protocol to:

Tissue processingThe ATPs are checked against maintenance checklist to ensure that all reagents are properly loaded and in sufficient amounts prior to start of processing. Reagents are maintained (after 20 cycles) to ensure a certain standard of quality of processing in subsequent cases.

EmbeddingParaffin dispensers are emptied monthly to clear debris collected at the bottom of the container. MicrotomyThe temperature of floatation baths are checked and recorded daily on the temperature log as part of equipment maintenance to ensure that it is in top condition during production of results. If out of range (less than 44oC or more than 50oC), corrective actions are taken by adjusting control knob. The microtomes and floatation baths are cleaned daily to remove debris after all sectioning done.

StainingWorking thermometer and equipment temperature display are calibrated monthly to make sure that readings displayed on ovens and refrigerators are within control range. The coldroom used to store special staining kits is inspected daily to ensure that it can function properly. The accuracy of its temperature is checked daily as the activity of special stains will be very much affected by temperature. When preparing staining reagents, the electronic balance may be used to measure out certain ingredients. Therefore, it is calibrated yearly to make sure the reading is accurate and within 1% of the reference mass as certain special stains are sensitive to quantity. Control samples are stained simultaneously with patients’ samples and observed for its quality. This is to ensure that any poor staining caused by defective reagents detected by control samples can be corrected at first hand before the sections are stained. H&E and special stains controls are evaluated and graded according to their quality from 5 (excellent) to 2 and below (inadequate in which remedial actions are to be taken). For H&E control, the nuclear and cytoplasmic control must be well defined and contrasted. As for special stains controls, there must be relatively good demonstration of 75% or more of the cells without background staining. During staining of patients’ samples, 10% of the slides contained in a rack are sent for microscopic examination for grading as part of QA. The slides are evaluated to detect any faults in staining, sectioning and mounting. Percentage of unsatisfactory slides caused by errors in staining, sectioning or mounting are counted monthly as well. Weekly, at the end of every Tuesday and Friday, reagents used for routine H&E are changed as part of QC so that the quality of staining can be consistent throughout. Records are maintained which indicate the type of reagent that is changed, filtered or replaced. Since Haematoxylin and eosin are prepared once a week, they are filtered daily to remove debris left by the previous slides. Before sending the slides for mounting, the machine is primed to remove any air bubbles present as it can affect the microscopic examination and thus, the quality of staining.

Dispatch slidesAll slides are checked against tissue blocks prior to their dispatching. If unsatisfactory, they are sent for recuts. Also, the number of slides indicated on request forms must tally against the amount being stained.

Lastly, pathologists evaluate the results in areas of sectioning, staining and mounting as these are the criteria that will affect the quality of section and thus, hinder diagnosis. The total number of errors that are being raised in all stages of workflow (from processing to labeling) are counted monthly and systematically documented in an ‘EVENTS’ file. In addition, a maintenance program for all equipment and instruments, listing the model and serial number, dates of services performed, parts installed, and the interval and date of the next service are kept and recorded. There is also a complete equipment inventory, listing the name and manufacturer of the instrument, model and serial number, purchased from, personnel who installed the instrument, the date, length of warranty, purchase price , and location of the manual of instructions covering the equipment.

Sunday, October 28, 2007

Alright, this week i'll be sharing more on the analysis, excision and identification of proteins. Also, i'll touch a bit on the DRP group that me and Jiaxin were put in charge of. This weeks blog is more of a reflective entry rather than a factual one.

After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.

Protein spot analysisAfter extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.

For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.

Protein spot excisionThe protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.

After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.

After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.

DRP studentsFor our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.

In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.

Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.

Saturday, October 20, 2007

heya.......first and foremost i would like to wish all muslim frenz SELAMAT HARI RAYA.......dun forget to collect as many green packets as possible..it might be our last chance...hehehe....aniwei this week i would post something that was shown to me on the 1st day of work......ISOLATION OF PLASMA AND WBC..yah i noe..a bit outdated....

Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....

This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......

Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....

A) Isolation of plasma1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min-this separates the plasma from the other blood components2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.-pipette as much supernatant as possible but do not disturb the pellet3. Store the eppendorf tubes at -80oC fridge**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)

B) Isolation of WBC1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes-the pellet is actually RBC2. Add 9ml of RBC lysis solution to the tubes-this forms 1:3 blood to lysis solution ratio3. Incubate the tubes at room temperature for 10 min4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min5. Pour away the supernatant6. Add 1ml of RBC lysis solution and pipette up and down7. Transfer everything from the tubes to eppendorf tubes8.Centrifuge the tubes at 3000rpm for 3min at 4oC9.Centrifuge at 15000rpm for 5 min at 4oCDiscard the supernatant11. Store the pellet (WBC) at -80oC fridge**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....

tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D

Wednesday, October 17, 2007

Gomennasai, for the late posting! Here's my posting for week 16th. Basically, for the past few weeks I have been involved in the statistics analysis for both of my gene projects using the SPSS software. SPSS is the abbreviation used for Statistical Package for the Social Sciences; it provides almost everything and anything you required to perform an analysis.

Common Statistics Perform in SPSSIn SPSS, it comprises of multiple statistics tests, most of them are covered in year 1 math stats module. Two of the more commonly features used are the descriptive statistics and the bivariate/multivariate statistics.

Descriptive statisticsThe most fundamental and frequent statistic feature used. It summarized the samples’ results and portrays the tabulated results in forms of graphs or tables. Thence it is mainly used for quick and basic analysis. Here are some of the statistics tests used in descriptive statistics:FrequenciesA simple measurement used to computes/determine the mean, median, modes and SD of the results (variables).

Cross tabulationA cross tab provide information on 2 or more variables’ distribution consecutively and is usually presents a table format. Therefore, a cross tab is different from a frequency test. Some of the statistics tests used abide by with cross tab are chi-square (which test for clinical significant in variables), contingency coefficient (which test for strong interaction between variables) and phi coefficient (which test for degree of interaction between variables).

Bivariate/Multivariate StatisticsAs the name implied, this feature is only suitable for 2 variables. Any statistics involving more variables would be under the multivariate statistics. Similarly here is a list of the some of the statistics tests used in bivariate statistics:

t-testCommonly used when the sample size is minute or when the standard deviation is unknown. It is used to test if a null hypothesis is true or false (accept or reject). For example, if null hypothesis used is: mean of A = mean of B (where both the means of the variables is equal). After a series of calculation if t < a =" mean">

ANOVAAn ANOVA test compares the variables’ mean with their variances. Unlike t-test, ANOVA used variances instead of t value and also assess on more than two variables together.

So how and wat statistics should be applied to different data analysis?The answer would depend on wat type of “story “ you wish to express in your research. For instance, if you wish to find out the relationship of drug X on blood glucose level. Then a linear regression approach would be an ideal.

A linear regression is a technique used to determine the relationship of dependent variables with independent variables. In this case, blood glucose level is your dependent variable while the dosage of drug X would be the independent variables.Once you identify your variables you can start analyzed using both the descriptive statistics and the bivariate/multivariate statistics.

Another technique used for data analysis is called the factors analysis. This approach look into dependent variables, meanwhile, indirectly identifies the independent variables within. For example, by looking at blood glucose level (dependent variables), other independent variable such as concentration of drug X would be determined.

Lost in this statistics info?? Don’t worry maybe these definitions can help out.Dependent variablesA dependent variable is an outcome, a prediction that can be influenced by the independent variable. Usually is something, which cannot be control.

Independent variablesAn independent variable is something, which you may control or predict in experiment.

MeansMean equal average

Null hypothesisA null hypothesis is a presumed statement before statistics analysisData analysisFor SPSS to analysis a data/ samples results, a syntax is often used. Simply type in the code (instructions) you want the SPSS to do and select run. Subsequently, the results will appear in the output file in a graph, table, histogram formats (etc). Based on the tabulated results, interpretations can be made.

That all for this week!! Stay tune to next week SIP sharing!!If you have any question on my post feel free to leave comment.

Monday, October 1, 2007

Anyway, I am currently extracting RNA from Formalin-fixed, Paraffin-embedded blocks. Hence, I shall talk about this process today yup!

Reason for extraction/isolation of RNA : Using this RNA extracted from the blocks, I will carry out Reverse-Transcription PCR to reverse-transcribe the mRNA I have gotten into cDNA and then carry out Real-time PCR to look at the genetic expression of a particular gene I am interested in. For more info on Real-time PCR, do refer to my group member, Avery’s blog entry (July 22, 07) as I believe she had written a very detailed entry about this technique =))

RNA isolation must be carried out over 2 days.DAY 1 :(Sectioning)

Using the microtome, I will section about 45um of tissue sections into a eppendorf tube.

(Deparaffinissation)

Add 800uL of xylene into the tube and mix.Significance: Since I am using FFPE blocks, I will first have to remove the wax from the section .

Blot the tube briefly onto a paper towel to get rid of the ethanol residues.

Dry the tissue pellet for 10mins at 55ºC

(Overnight Proteinase K Digestion Incubation)

Add 100uL of Tissue Lysis Buffer, 16uL 10% SDS and 40uL proteinase K into the dried tubes and vortex briefly before incubating the tube overnight at 55ºC.Significance: This is to disrupt the protein structures in order to get the mRNA we want

DAY 2:

(RNA isolation)

Add 325uL of binding buffer and abs ethanol to the tube and pipette to mix.

Significance: Binding buffer will bind to the nucleic acids we want and allow the contaminants to flow through the filter and into the collection for discard.

Combine the filter tube and collection tube and pipette the lysate into the upper reservoir

Significance: The nucleic acids will bound to the chaotropic salts specifically to the surface of the glass fibers pre-packed in the filter tube while all other contaminants (salts, proteins and other cellular contaminants) will be washed off the column.

Centrifuge the High Pure filter for 2 mins at max speed.

Place the High Pure filter tube into a fresh 1.5ml reaction tube, add 90uL Elution Buffer and centrifuge for 1 min @ 8000rpm before collecting the flowthrough.

Add 10uL DNase Incubation Buffer and 1.0uL DNase I to the eluate and mix. Incubate for 45mins at 37 ºC

Significance: DNase I will help to remove any residual DNA since all we want is the RNA from the blocks.

Significance: A second incubation step with Proteinase K is to improve on the quality of the RNA as well as to ensure that all protein structures are disrupted.

Repeat the steps involved for RNA ISOLATION until the part in which the High Pure filter is centrifuged at max speed for 2 mins.

This time round, add only 50uL of Elution Buffer and centrifuge for 1 min@ 8000rpm.

Using the NanoDrop spectrophotometer, we can then check the RNA concentration of the 50uL eluate we obtained from our RNA extraction! =)

All the reagents/buffer were from this kit known as High Pure RNA Paraffin Kit from Roche, Switzerland. Therefore, i do not know the content of some of the reagents such as the Wash Buffer I and II which is why i cannot specifically tell you guys what each and every one of the reagents does ya. Hope this is alright with you guys. But i have tried to explain as many steps as possible so the whole idea behind this RNA isolation process should be pretty clear =)

(1) During sectioning, the tissue blocks are constantly exposed to the existence of RNase in the surrounding. Hence, the RNA in these blocks might risk getting destroyed during the sectioning process and affecting the total concentration of the RNA after the RNA extraction is done by the end of Day 2.(2) The process of fixing the tissue sample and embedding it in paraffin has caused severe degradation of the RNA. Therefore, it’s more difficult to isolate good quality RNA from FFPE tissues.(3) The “older” FFPE blocks tend to give poorer RNA concentration as compared to the tissues that were fixed only recently.

Ways to overcome these difficulties:

(1) Use RNase Zap to help reduce the amount of RNase that could be present in the surrounding (microtome/gloves). If talking is necessary during the isolation process, one could wear facemask so as to prevent the RNase in the saliva from degrading the samples.(2) Since it’s more difficult to isolate RNA from FFPE tissues, more sections could be collected in several tubes and pooled together so as to maximize the concentration of RNA to be collected after 2 days of extraction.(3) If given a choice, choose tissue blocks that were fixed and embedded recently (within 5 yrs) because studies have found that RNA in the blocks that were more than 10 year-old degrades significantly.

Saturday, September 29, 2007

Hey..this week’s my turn, and I have interesting PICS to share from my research work.

As I have mentioned before, my project is basically delivering DNAs into cancer cells using synthetic carriers such as polymers for future gene therapies. Hence, I’m doing the last part of my project which is ‘localisation of polymer-DNA complexes’ in the cells.

For the localization studies, my main aim is to determine whether my polymer goes into the nucleus. Hence I have to label my polymer, nucleus and DNA. However, since my DNA labeling kit has not arrived, and I want to train myself using the confocal microscope, I started my experiment first using my labeled polymer and nuclear labeling.

The cell line I chose is HepG2, it is a human hepatocellular carcinoma cells, mainly because it does not detached easily from the tissue culture plate. I label my polymer with FITC, and to stain the nuclei, I used Hoechst dye that is specially used to stain ‘live’ cells.

Why use CLSM?

In order to view my cells, Confocal laser scanning microscopy(CLSM) is used. The CLSM used is LSM 5 LIVE, the latest edition for imaging of living cells from ZEISS technology. CLSM uses laser to excite the fluorochromes, unlike fluorescence microscope(FM) which uses fluorescence light.

1)CLSM allows 3D view of the sample, hence if there is an overlapping of different fluorescent signals, only CLSM can help differenciate them.

2) CLSMallows viewing of distinct and sharp images by removing out-of-focus light, unlike FM that usually gives blurry images and some signals cannot be viewed distinctly.

If you wanna know the principle of CLSM and FM, just give me a comment, as it is more like ‘physics’, afraid some of you might not understand.

Ok back to my experiment, I prepared 4 different samples at different time interval: 1, 2, 3 and 4.5 hr, these time intervals are chosen after reading some science papers and also FITC ‘photobleaches’ easily, hence I have to prepare samples at different time intervals. The polymer-DNA complexes are given(transfected) to the cells at 0 hr, while the nuclei are stained with Hoechst 40 mins before viewing under CLSM, and the cells are washed 5 times with colourless DMEM medium before viewing to prevent auto-fluorescence. Hence, that is why i used HepG2, because of the washing steps.:)

One important precaution: sample preparations must be done in the dark, once exposed to light, the fluoresce intensity can greatly decrease, especially for FITC.

GREEN(FITC): polymer ( note: DNA is still bound to the polymer, but cannot be seen as it is not labeled)

BLUE(HOECHST): nucleus

The following pictures are a bit blurry because I magnify them 8-12 times from the original one.

1 hr after Transfection: on the cell membrane

Q: Why do the polymers 'stick' onto the membrane?

A: My polymers are cationic(+vely charged), and the cell membrane has proteoglycans(-vely charged), hence the ATTRACTION!

2.5 hr after Transfection: in the cytoplasm

Q: How does my polymer enters the cytoplasm?

A: Based on other science papers, it is known that polymer-DNA complexes enter the cells via endocytosis. However the actual mechanism(type of endocytosis) is still a BIG question, and no one knows.

4.5 hr after transfection: in the nucleus!!

Q: How can the polymers(size: 100-200nm) go into the nucleus, when nuclear pores only allow molecules of less than 10 nm to pass through?

A: This is also a another BIG question that many scientists are still finding. However based on latest papers, it is known that cationic polymers do interact with the anionic phospholipids on the cell membrane and eventually coat the polymer-DNA complexes. These coated complexes then fuse with the nulear envelope, and release the complexes.

Anyway, frankly, i hate 'Live' confocal studies, because it is very tedious, and u will spend the entire day on it, plus TIME is a GREAT factor. Also usually, there will be cells that do not show similiar pattern like others under the microcope, hence, I have to search and search. :( But still, research is FUN! :))))

Observations of prostate trimming by pathologists-Material: Prostate cutting tool-Whole mount is necessary as tumour is located in prostate and can only be seen under microscope.-For whole mount processing, the timing of fixation will be extended for 41/2hours (total 211/2 hours) to ensure tissue is thoroughly fixed.-The dehydration steps will also be extended to ensure proper dehydration due to the size of the tissue.-Other tissues will not be processed together with whole mount prostate as over fixation and dehydration of smaller pieces will cause the tissues to be harder.

Microtomy

1. Microtome safety (Safe Operating Procedure)2. Handle blades very carefully when installing or removing. Follow the manufacturer’s guidelines explicitly.3. Tungsten-Carbide knives can put through your shoes, if dropped. Be careful where your feet are positioned when installing or removing blades.4. Store blades in a covered container that has guides to hold the blades rigid.5. Never leave blades on countertops. Lacerations can occur when reaching across the countertop and inadvertently contacting on unprotected table.6. When setting up the microtome, position the sample first, and then put in the blade. Never the other way around.7. When applying the brake, ensure that it is tight. Most accidents occur when the brake slips and the operator’s hand is drawn into the blade.8. When leaving the microtome, even for a short time, ensure that the blade guard is in place.9. When preparing a paraffin sample for the microtome, remember to clamp the sample down tight. The movement is allowed by a loose clamp increase your risks of cut.10. Use forceps or brushes during retrieving and transferring slides and ribbons from the blade, thereby keeping your hands free from the moving parts of the microtome.

Tissues turn hard and cut like stone1. After clearing, the specimen was / 1. Nothing can be done except repeataccidentally left to dry in air with fresh specimen2. Paraffin bath is too hot and burns the tissue / 2. Decrease the temperature of parrafinbath

Split ribbon or lengthwise scratches in ribbon1. Nicks in the blade / 1. Use a different part of the blade.2. The tilt of the knife is too great / 2. Decrease the tilt3. Blade edge dirty / 3. Wipe blade edge with xylene4. Hard particles (calcified materials) in the block / 4. Do surface decalcification5. Crystal from fixative e.g. Mercuric chloride / 5. Treat tissues to remove crystals

Undulations in the surface of the section1. The blade is not tightened / 1. Tighten all screws2. The title of the blade is too great / 2. Decrease tilt to prevent vibration3. Tissue too hard e.g. cervix, fibroid / 3. Soften tissue in phenol or mollifex4. Blade edge is dull or rounded / 4. Change to new blade edge

Sunday, September 16, 2007

Hey guys! 12 weeks have passed. I have started with a second project that covers expression proteomics of a clinical isolate of Stenotrophomonas maltophilia, a nosocomial infectious agent. Expression proteomics relates to studying the entire secretory profile of the bacteria. In my case, i will be precipitating the secretory proteins from S. maltophilia grown at both 28˚C and 37˚C.

Methods1) Isolate is streaked on an LB agar plate and incubated at 37˚C for 24 hours2) A colony will be inoculated into 20mL of LB broth and incubated at 28˚C for 24 hours3) 5x10^7 cells will be inoculated into 12 tubes in which 6 will be incubated at 28˚C and theother 6 will be incubated at 37˚C4) Secretory proteins in the supernatant after centrifugation will be transfered to teflon tubes5) Equal volumes of 40% TCA in Acetone as the supernatant will be added to precipitate theproteins6) Tubes will be incubated at 4˚C for 1 hour and inverted every 10 mins7) Tubes are then centrifuged at 16,000xg for 1 hour at 4˚C8) Keeping an eye on the protein pellet, supernatant is decanted9)Remaining supernatant is aspirated out and 250uL of pre-chilled Acetone is added10) Acetone is used to wash the proteins11) Washed protein is transfered to a fresh Eppendorf tube and centrifuged12) Supernatant is aspirated and pellet is allowed to air-dry13) 200uL of Rehydration Buffer is used to rehydrate the protein pellet14) 2uL of Protease inhibitor was added to prevent denaturation of proteins15) Bradford Assay is performed to determine the final protein concentration16) If there is sufficient protein sample, a 2-Dimensional gel can be run17) Spots containing significant proteins can be identified and excised18) Protein spots are then analyzed through the MALDI TOF/TOF19) Peptides can then b identified through the Mascot Search program

ChemicalsThat was a brief itinery of what is carried out in my experiments daily. A single experiment can last for up to 2 weeks. Now i'll explain about certain chemicals that are required for protein precipitation.

Acetone is used for the washing of protein pellets after precipitating with 40% TCA in Acetone. Acetone is the simplest and most important of the ketones. It is a polar organic solvent and therefore dissolves a wide variety of substances. It has low chemical reactivity. These traits, and its relatively low cost, make it the solvent of choice for many processes. About 25% of the acetone produced is used directly as a solvent. Acetone is also used as a drying agent, due to the readiness with which it binds to water, and its volatility. Also, by washing with Acetone, any impurities that have accumulated in the process is removed from the proteins.

Since Acetone can act as a drying agent, the protein pellet will be rid of most liquids thus Rehydration Buffer is required to rehydrate the proteins. The Protease Inhibitor is used to prevent any proteases from acting on the precipitated proteins. Proteases may still be present because they ar enzymes which are also proteins.

Aseptic techniquesWhen working with proteins to perform analysis, it is important to not come into contact with the samples since it can contribute to the quality and quantity of proteins analyzed. For example, if we do not wear gloves and come into contact with the sample, keratin may be found abundantly in our protein sample. If we talk into our tubes during air-drying, enzymes may be added to our samples.

ConclusionThus, i conclude by saying that even though research may seem easy to some and boring to others, i have to say that they are very wrong. I have enjoyed my 12 weeks at the research centre so far through all the ups and downs and all the "failed" experiments. It is inevitable not to get good results but they ARE results so we learn from them. This is the end of my second entry after 12 weeks. Hope to blog in 6 weeks time with more interesting facts to share. See you guys at the next Campus Discussion!

Sunday, September 9, 2007

Heya guys....how are all of u doing......???11 weeks have passed.......9 more weeks to go...dunnoe if that's good news or bad news....8-?......ahkahkahk....aniwei this time round....i would be blogging about something all of us are very familiar with.......PCR!!!.......

Title: PCR of subjects' DNA (24 for Chinese, Malays and Indians each)

Purpose: To make a lot of copies of the specific DNA fragment that needs to be analysed.